Construction Of Recombinant Baculovirus With CBA Promoter EGFP

Many thousands of recombinant proteins have been successfully produced in baculovirus-infected insect cells and larvae. Baculovirus fails to replicate in mammalian cells, but it does express alien genes under the control of appropriate promoter used to drive the expression of foreign genes. Easy to manipulate and large DNA fregments containing, coupled with its non-toxic and non-replication nature, makes baculovirus a very useful tool for studying genetically engineered vaccine. Researches on enhancing recombinant baculovirus infecting efficiency and prolonging expression of transgenes have become more and more important. In this thesis, two types recombinant baculovirus containing CBA promoter(Complex promoter contains CMV ie enhancer region and Chicken actin promoter region) expression cassette were constructed, and established a new way for the researchment of genetically engineered vaccine.In this thesis, two transfer vector were constracted based on pFastBac1 plasmid vector eliminating pPolH promoter. An expression cassette including VSV-GED(Truncated vesicular stomatitis virus G protein) under the control of pPolH promoter was inserted into the vector aims at enhancing the transfection efficiency. Another expression cassette drivered by CBA promoter was inserted into the same vector in the opposite direction of pPolH promoter, including WPRE (wood-chuck hepatitis virus post-transcriptional regulatory element) in the 3’-UTR of EGFP gene, which was a post-transcriptional regulatory element can boost baculovirus-mediated gene expression in vertebrate cells. Then the first transfer vector pSD-EGFP was finished. And then construction of another transfer vector pSD-ITRs-EGFP was achieved by flanking ITRs(Adeno-associated virus inverted terminal repeats) to the CBA-EGFP-WPRE expression cassette in order to prolong the expression of transgene. After getting the restriction map of the two transfer vectors, recombinant transfer vectors pSD-EGFP and pSD-ITRs-EGFP were transformed into E.coli DH10Bac competent cells to extract recombinant bacmid DNA for transfecting Sf9 insect cells in order to acquire high titer recombinant baculovirus. The expression of EGFP gene was greatly enhanced at 72 hours after MOY cells were infected by recombinant viruses. In this research, viral pseudotyping and adding regulatory element into baculovirus genome were both employed to enhance the gene transfer efficiency. And this will facilitate with the researching of baculovirus-based genetically engineered vaccine.